Prevalence and Association of Human Papillomavirus with Esophageal Squamous Cell Carcinoma in Iran: A Systematic Review and Meta-Analysis.

Background: Human papillomavirus (HPV) can infect the epithelium of the esophagus, but so far there is no reliable and comprehensive evidence about the prevalence and association of HPV with esophageal cancer in Iran, as high incidence region. This study aimed to evaluate the prevalence and association of HPV with esophageal squamous cell carcinoma (ESCC) in Iran. Methods: Relevant English and Persian articles published up to Aug 2017 and indexed in databases were reviewed. Frequency of HPV genotypes in ESCC cases and controls was surveyed according to regions of Iran. Data were meta-analyzed with random effects models using Comprehensive Meta-Analysis software. Results: Overall, 14 studies were eligible including 1444 samples (1062 ESCC cases and 382 controls). HPV was positive in 269 (25.32%) of 1062 ESCC cases and in 65 (17.01%) of 382 controls. Total prevalence of HPV in both groups was estimated 0.256 (95%CI, 0.208%–0.310%). The prevalence of HPV-16 and HPV-18 was estimated 0.121 (95%CI: 0.087–0.183) and 0.046 (95%CI; 0.023–0.088), respectively. The difference in HPV prevalence in different regions of Iran was statistically significant (Q=18.20, df =4, P=0.001). In 6 case-control studies, the pooled odds ratio was estimated 1.99 (95%CI; 0.916–4.315). Conclusion: High-risk HPVs were observed in ESCC cases and controls from different regions of Iran. The odds ratio indicates that the HPV infection in ESCC cases was approximately 2 fold more than the controls. More case-control studies in other populations with larger sample size are necessary.


Introduction
Esophageal cancer (EC) is the most offensive malignancy of the gastrointestinal system and the eighth most common cancer worldwide (1). EC histologically has two subtypes, esophageal adenocarcinoma (EAC), and esophageal squamous cell carcinoma (ESCC). They vary in geographical distribution and etiology. ESCC is prevalent in developing countries (2,3). High incidence of EC areas, followed by Mazandaran and Khorasan Provinces, all in northeastern of Iran. The incidence of EC from 2005 to 2006 was 5.83 and 6.25 in males and females, respectively. Compatible with other areas of the world, ESCC constitutes more than 90% of all EC in northeastern of Iran (6,7). EC is commonly diagnosed late when tumors have invaded peripheral tissues leading to dysphagia. Although, there are therapies for EC such as surgery and chemoradiation, prognosis of EC is weak. Nowadays, there are no screening methods to detect EC at early stages. Thus, recognition of etiology and applying preventive method is very important to control EC (2). The probable cause of the EC consists of several risk factors that cooperate in a multi-stage process, including alcohol consuming, tobacco smoking, nutrition deficiencies, and infectious agents (1). In addition, some chemicals and lifestyle factors, including polycyclic aromatic hydrocarbons, opium use, hot tea drinking and genetic factors (oncogenes, tumor suppressor genes, cell cycle regulatory proteins) were associated with EC in north of Iran (2). As a potential etiology of EC, that has been reported recently was human papillomavirus (HPV) infections, especially with high-risk genotypes including HPV-16 and HPV-18. The evidence of this relationship was weak and the results of these studies are not consistent (1). However, HPV infection is really associated with cervical, vaginal, vulvar, anal, penile, head and neck cancers (8). The genome of HPV is a double-stranded circular DNA including early (E1 to E7) and late (L1, L2) genes or open reading frames (ORFs) encoding viral nonstructural regulatory proteins and viral structural proteins, respectively. Carcinogenesis of HPV is related to E6 and E7 which deregulate the cellular regulatory proteins (p53 and Rb), therefore leading to proliferation of the host cell. However, E2 has suppression effect on the E6 and E7 promoters. The genome of HPV integrates into the host cell chromosome in malignant tissues and disrupts E2 ORF. Therefore, decreased expression of E2 regulatory protein results in an increased expression of the viral E6 and E7 oncoproteins (9).
Using the sequence of L1 gene, about 200 HPV types have recognized. High risk HPV types including HPV-16 and 18 are more frequent in cancerous tissues, but other types [30, 31, 33, 35, 39, 45, 51-53, 56, 58, 59, 66, 68, 73, 82] are less associated with cancers (10). Some meta-analyses were conducted to estimate the prevalence and association of HPV with ESCC worldwide (5,11,12), but Persian language papers were not included in them. In addition, molecular studies were conducted about the HPV effect on ESCC in some regions of Iran (4,(13)(14)(15). However, there is no reliable and comprehensive evidence about the prevalence and association of HPV with esophageal cancer in Iran, as high incidence region. We aimed to perform a systematic review and metaanalysis to determine the prevalence and association of HPV with esophageal cancer in Iran.

Literature search
All studies conducted on the prevalence of HPV in esophageal cancer in Iran were searched systematically from 2002 up to Aug 2017 for relevant articles published in English or Persian languages and indexed in databases such as PubMed, Google Scholar, Magiran, IranMedex, and Iranian Scientific Information Databank (SID). Keywords were "Human Papillomavirus", "HPV", "Esophageal Cancer", "Esophageal Squamous Cell Carcinoma", "ESCC" and "Iran". Bibliographies and full texts of studies reporting HPV prevalence in esophageal cancers in Iran were entered into the EndNote software, according to the STROBE Statement-Checklist.

Inclusion criteria
1. Studies reporting HPV infection in esophageal cancer and noncancerous tissues in Iran were selected; 2. only full-text studies in English or Persian articles were included.

Exclusion criteria
1. Studies that the sampling location was unknown; 2. Studies that do not clearly outline the data; 3. Case report/series; 4. Letter to the editors; 5. Review articles; 6. Abstracts of conferences; 7. Studies that sampling took place at the same time and place reporting the same data.

Selection and Data Extraction
Studies were identified based on the inclusion and the exclusion criteria from all English and Persian language articles reporting the frequency of HPV or its genotypes in esophageal cancers and controls in Iran. All appropriate articles were evaluated by two researchers, independently. With the exclusion of inappropriate studies, the data extracted and entered into an Excel file. If multiple publications reported the same data from the same population, only the publication with newer data was included. Extracted information were including first author, study location (province/city), publication year, if available dates of sample collection, sample size, gender, specimen type, frequency of esophageal cancers (cases), frequency of non-cancerous esophageal tissues (controls), HPV detection method, HPV genotyping method, overall and type-specific frequency of HPV in esophageal cancers (cases), or in non-cancerous esophageal tissues (controls), and grade of malignancy. For studies with samples from multiple province/city, data were extracted separately if possible.

Statistical analysis
Data were analyzed using CMA (Comprehensive Meta-Analysis 2.2.064) software. The effect size was calculated as event rates and their 95% confidence interval. At first, the heterogeneity of the studies was evaluated using the Cochran's Q and I 2 indexes. The homogeneity was rejected; we used the random-effects model to analyze the publication bias using the Egger test and graph. To estimate the prevalence of HPV genotypes based on age, year of publication, and geographical area, metaregression was used. At all steps, P-values below than 0.05 were considered statistically significant.

Results
Based on the preliminary search strategies 261 studies were identified through database search-ing up to Aug 2017. In addition, four studies were found manually. From 265 studies screened, 248 studies were excluded, including unrelated articles, reviews, case reports, abstracts of conferences, and articles with incomplete information, based on the inclusion and exclusion criteria. Finally, 14 studies were eligible and were included in the meta-analysis (Fig. 1). Studies were categorized into five geographical regions of the country including Center (Tehran Province), North (Guilan, Mazandaran and Golestan provinces), South (Fars Province), East (Khorasan Razavi Province) and West (Kermanshah and Kurdistan provinces) ( Table 1). Total sample size in 14 studies was 1444, including 1062 esophageal squamous cell carcinomas (ESCC) cases, and 382 esophageal non-cancerous tissues (controls). Esophageal non-cancerous tissues had been taken from patients with dysphagia, endoscopically normal or margins of carcinomas. Specimen type of all of the samples had been biopsies from esophageal tissues, as formalin fixed and paraffin embedded (FFPE) tissue blocks. In the studies that reported the age of patients, the age ranged from 22 to 91 yr in ESCC cases, and from 22 to 90 yr in the controls. In the studies that reported the gender of patients with ESCC; out of 1005 patients, 578 were men, and 427 were women; only one study (16) had not reported the gender of patients. Five studies reported the gender of non-cancerous patients, in which out of 192 patients, 95 were men and 97 were women, because of space restriction data were not shown in Table 1. The laboratory test used for DNA suitability in all of the studies was polymerase chain reaction (PCR) for the host cell beta-globin gene. In all studies, PCR had been conducted for detection of HPV using standard primers designed for viral L1 gene (GP5 + /GP6 + and MY09/MY11), and E6 and E7 gene sequences. The laboratory methods used for detection of HPV genotypes were genotype specific PCR primers (E6/E7), sequencing of the PCR product, Real-time fluorescence detection (FRT) kit, restriction fragment length polymorphism (RFLP), and INNO-LiPA HPV genotyping kit (Table 1).  (Table 1). According to the values of I 2 =87.79, and P<0.001, we used random effect model in meta-analysis, thus the pooled overall prevalence of HPV was estimated 0.256 (95% CI, 0.208%-0.310%) in both groups (Fig. 2). The difference in HPV prevalence in different regions of Iran was statistically significant (Q=18.20, df=4, P=0.001). According to regions of Iran, the maximum prevalence of HPV infection was 0.378 (95%CI, 0.293%-0.472%) in North of Iran (Guilan, Mazandaran, Golestan, Turkmen Sahra), as high incidence region (Fig.  2). Moreover, according to the value of I 2 = 80.25 and P<0.001, we used the random effects model to estimate the overall prevalence of HPV, according to publication years. There was a decreasing trend in the overall prevalence of HPV, according to publication years (Fig. 3). However, there was an increasing trend in the overall prevalence of HPV according to patient's ages. The funnel plot showed that there was publication bias (Fig. 4). Only five studies had reported the frequency of HPV infection, according to gender of patients with ESCC. Therefore, HPV was positive in 65 men and in 50 women with ESCC. HPV genotypes had been reported separately or as combined infections in the most of the studies. Three studies had reported untypeable or unknown HPV genotypes. However, the genotypes of HPV in ESCC cases and controls according to each study are shown in Table 1. Most of them were high-risk HPV types. Total HPV genotypes are shown in the last row of Table 1.  (2), HPV-6/16 (2), HPV-6/18 (1), HPV-6 (1), untypeable (3) -    The pooled prevalence of HPV-18 was estimated at 0.045 (95%CI; 0.023-0.088) by using the random effect model (P<0.001, I 2 =58.368) (Fig. 6).
In addition, there was publication bias about HPV-18. The prevalence of HPV-18 was decreasing according to publication years and with the ages of patients. The difference of HPV-18 prevalence in different regions of Iran was not statistically significant (Q=5.43, df=2, P=0.07). Non-cancerous controls had been included in 8 of the reviewed studies; but, the frequency of HPV in ESCC cases was zero in two studies (Table 1). Therefore, only 6 studies were analyzed as casecontrol. According to the values of I 2 =75.55 and P =0.001, we used the random-effects model. The estimated pooled odds ratio (OR) was 1.988 (95%CI; 0.916, 4.315), which indicates that the exposure ratio in ESCC cases was approximately two-fold more than the controls. However, this ratio was not statistically significant with respect to the confidence interval (P=0.08) (Fig. 7). Overall, 8 studies had reported the histopathological degree of ESCC in the patients, including 141 well differentiated, 182 moderately differentiated, 91 poorly differentiated, and 33 not differentiated. The histopathological degree of ESCC cases was not statistically significant with respect to study regions, years of publication and ages of patients.   (31).
A study reported the prevalence of HPV in oral of healthy individuals and relative risk factors in Iran. Saliva samples of 114 healthy individuals were collected for HPV DNA detection. The frequency of oral HPV was 7 (6.1%) in healthy individuals. Virus type in five of them was HPV-18. Moreover, HPV-6 and HPV-66 types were detected in two cases. Relation of oral HPV positivity to demographic data and risk factors was not statistically significant (32). Power of association between HPV and esophageal cancer has not been as strong as detected in cervical and laryngeal cancer (28). This may be due to the small number of appropriately designed studies (3), such as case-control studies with large sample sizes (5).
In comparison, the results of HPV prevalence and its association with ESCC in the present study were somewhat similar to some previous studies. The differences of results of in the present study may be due to geographical region, environmental factors, differences of the population (race, living habits), research design, sample size, type of specimen, statistical analysis, and heterogeneity of included studies.

Conclusion
High-risk human papillomaviruses were observed in ESCC tissues and controls from different re-gions of Iran. The odds ratio indicates that the HPV in ESCC cases was approximately two-fold more than the controls. With respect to the defined oncogenic role of HPV in some cancers such as cervical cancers, the causality of this virus in ESCC needs more case-control studies in other populations with larger sample size.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.